Abstract
Previous studies have shown that glucagon cooperatively interacts with insulin to stimulate hepatic FGF21 gene expression. Here we investigated the mechanism by which glucagon and insulin increased FGF21 gene transcription in primary hepatocyte cultures. Transfection analyses demonstrated that glucagon plus insulin induction of FGF21 transcription was conferred by two activating transcription factor 4 (ATF4) binding sites in the FGF21 gene. Glucagon plus insulin stimulated a 5-fold increase in ATF4 protein abundance, and knockdown of ATF4 expression suppressed the ability of glucagon plus insulin to increase FGF21 expression. In hepatocytes incubated in the presence of insulin, treatment with a PKA-selective agonist mimicked the ability of glucagon to stimulate ATF4 and FGF21 expression. Inhibition of PKA, PI3K, Akt, and mammalian target of rapamycin complex 1 (mTORC1) suppressed the ability of glucagon plus insulin to stimulate ATF4 and FGF21 expression. Additional analyses demonstrated that chenodeoxycholic acid (CDCA) induced a 6-fold increase in ATF4 expression and that knockdown of ATF4 expression suppressed the ability of CDCA to increase FGF21 gene expression. CDCA increased the phosphorylation of eIF2α, and inhibition of eIF2α signaling activity suppressed CDCA regulation of ATF4 and FGF21 expression. These results demonstrate that glucagon plus insulin increases FGF21 transcription by stimulating ATF4 expression and that activation of cAMP/PKA and PI3K/Akt/mTORC1 mediates the effect of glucagon plus insulin on ATF4 expression. These results also demonstrate that CDCA regulation of FGF21 transcription is mediated at least partially by an eIF2α-dependent increase in ATF4 expression.
Highlights
FGF21 is an atypical member of the fibroblast growth factor family that lacks the ability to bind to heparin sulfate proteoglycans, allowing it to escape the extracellular matrix and function in an endocrine manner [1,2,3]
We show that glucagon plus insulin and chenodeoxycholic acid (CDCA) increase the expression and DNA binding activity of the stress-associated transcription factor activating transcription factor 4 (ATF4) and that this protein plays a key role in mediating the stimulatory effect of these signaling factors on FGF21 gene transcription
Identification of Cis-acting Sequences That Mediate the Stimulatory Effect of Glucagon Plus Insulin on FGF21 Gene Transcription—To identify cis-acting sequences mediating the stimulatory effect of glucagon plus insulin on FGF21 gene expression, transient transfection experiments were performed in primary rat hepatocyte cultures using reporter constructs containing 5Ј deletions of the rat FGF21 promoter linked to the luciferase gene
Summary
FGF21 is an atypical member of the fibroblast growth factor family that lacks the ability to bind to heparin sulfate proteoglycans, allowing it to escape the extracellular matrix and function in an endocrine manner [1,2,3]. Ablation of the PPAR␣ gene suppresses the ability of starvation and ketogenic diet consumption to increase hepatic FGF21 mRNA abundance and serum FGF21 concentration [7, 8]. Another pathway mediating the nutritional regulation of FGF21 expression is activated by the glucagon receptor. This has been deduced from studies in mice showing that ablation of the glucagon receptor suppresses the ability of starvation to increase hepatic FGF21 mRNA abundance and serum FGF21 concentration [1]. The inability of glucagon to induce FGF21 mRNA abundance in hepatocyte cultures [14] contrasts with the results of gluca-
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